This invention relates to a germination inhibitor of spores, its production and a food using it to improve its keeping quality.
Heretofore, one of the most common methods for sterilizing food in its production is heating, such as pasteurization at a temperature less than about 100xc2x0 C. or high temperature sterilization at 100xc2x0 C. or more.
It is possible to sterilize microorganisms in a form of vegetative cell growing in usual circumstances, but bacterial spores exhibiting thermostability cannot be sterilized. While, foods sterilized at a low temperature are occasionally spoiled or putrefied by sporogenous bacteria generated by the germination of survived spores.
Contrarily, bacterial spores having ordinary thermostability can be sterilized by sterilization at a high temperature as well as growing microorganisms. Although high temperature sterilization makes bacterial spores harmless, it simultaneously allows foods to lose their nutrient components and to vary their properties and flavors. Thus, high temperature sterilization degrades food qualities.
In view of food storage, it is considered that the problem of bacterial spores resistant to heat sterilization can be solved by the control of germination of the spores. For examples, all spores are once germinated in advance to be made into a form of vegetative cell, and then sterilized. Alternatively, germination of all spores is inhibited to prohibit multiplication for a long period, or the like.
As to the former method, several spore germinators for Bacillus subtilis are known, such as a mixture of L-asparagine, D-glucose, D-fructose and KCl and L-alanine (Yoetsu Hachisuka, xe2x80x9cGahogaku (Sporologies)xe2x80x9d, Tokaidaigaku Shuppankai, 1988). However, this method is not so effective, because of the presence of spores inferior in germination reactivity remarkably, different spore germinators for the type of bacteria, or the like.
As to the latter method, there are known spore germination inhibitors for Bacillus subtilis, such as D-amino acids including D-alanine (Y. Yasuda and K. Tochikubo: Microbiol. Immunol., 29, 229 (1985)), various alcohols (Y. Yasudaxe2x80x94Yasaki, et al.: J. Bacteriol., 136, 484 (1979)), various fatty acids, etc. (T. Yasuda, et al.: J. Med. Chem., 25, 315 (1982), HgCl2, and so on, but this method is also not so effective, because of insufficient inhibition, remarkably high toxicity, or the like.
An object of the invention is to provide a spore germination inhibitor, which renders foods, etc. contaminated with thermostable bacterial spores as well as general bacteria, preservable by the sterilization in the degree of pasteurization without conventional high temperature sterilization which degrades food qualities.
Another object of the invention is to provide a method of producing the spore germination inhibitor.
Still another object of the invention is to provide a food using the spore germination inhibitor.
The inventors investigated eagerly in order to achieve the above objects, and concluded that the most preferable means is to use a natural germination inhibitor for bacterial spores. Thereupon, they further investigated in order to find a substance which inhibits germination of bacterial spores effectively, and as a result, they have found that bacteria belonging to genus Bacillus which are sporegenous, such as Bacillus subtilis, produce a spore germination-inhibiting substance which has a great spore germination-inhibiting ability and no toxicity problem, that the germination-inhibiting substance is 6-carbamoyl-2-pyridine carboxylic acid, and that its salts and derivatives also have germination-inhibiting action, to complete the invention.
Thus, the present invention relates to a spore germination inhibitor which comprises 6-carbamoyl-2-pyridine carboxylic acid or a salt or derivative thereof, a food containing it, and a method of producing 6-carbamoyl-2-pyridine carboxylic acid which comprises culturing a bacterium belonging to genus Bacillus, and collecting 6-carbamoyl-2-pyridine carboxylic acid from the culture medium.
The microorganisms belonging to genus Bacillus, which produce 6-carbamoyl-2-pyridine carboxylic acid (CPC), are not especially limited, and include Bacillus subtilis ATCC 33234, FERM BP-5325, FERM BP-5367, etc.
The form of culture may be either liquid culture or solid culture, and submerged culture with aeration is preferred in the industrial viewpoint. Nutrients in a culture medium may be conventional carbon source, nitrogen source, inorganic salts and the other minor nutrients used for culture of a microorganism. Culture is carried out under aerobic conditions at a temperature capable of growing the bacterium employed in the invention to produce CPC, preferably in the range from 30 to 40xc2x0 C., for a period from 5 hours to 2 days.
The spore germination inhibitor can be separated from a cultured matter by a conventional method for purifying a material, such as solvent extraction, various chromatographys, gel filtration or the like.
6-carbamoyl-2-pyridine carboxylic acid (CPC) which is the spore germination inhibitor of the invention has the following formula and properties. 
White powder
Soluble in water, particularly alkaline water, and organic solvent, such as pyridine and DMSO
The subject bacterial spores, which are inhibited by the spore gemination-inhibiting substance of the invention, are spores of sporogenous bacteria found in usual contamination of foods, such as Bacillus subtilis, Bacillus cereus, Clostridium perfringens and the like.
CPC used in the invention is not limited to the CPC produced by the aforementioned bacteria, and includes CPC produced by conversion with an enzyme or through chemical synthesis.
The salt of CPC may be any salt containing Na, K or the like or Cl, NO3 or the like.
The derivative of CPC includes CPC of which xe2x80x94CONH2 at 6-position is substituted by a functional group containing an amide bond represented by xe2x80x94CONHR. For example, effective derivatives are containing xe2x80x94CONHR wherein R is xe2x80x94NH2 or xe2x80x94NHR is an amino acid residue. Still effective derivatives are carboxyl group and/or carbamoyl group are transferred to any other position from 2-position to 6-position.
The salts and derivatives of CPC can be produced by a conventional method.
A suitable blending amount of CPC, its salt or derivative is 0.01 to 5 wt. % of a food to be stored, preferably 0.05 to 2 wt. %. CPC, its salt or derivative can be used as a germination inhibitor as is, but a known substance to inhibit germination, such as D-alanine, various alcohols or various fatty acids, may be combined therewith. Furthermore, a known fungicide, bacteriocide or preservative may be combined.
Spore germination-inhibiting effects are exhibited remarkably by combining D-alanine with CPC or its salt or derivative. Although several germination inducing substances, which stimulate germination of spores of Bacillus subtilis, are known, such as L-alanine, in addition to a mixture of L-asparagine, D-glucose, D-fructose and KCl (AGFK system), it is difficult to specify which component stimulates germination in the presence of many nutrients, such as in food. Therefore, as mentioned previously, it is expected that, although germination of spores in food mainly belonging to AGFK system can be inhibited by CPC alone, the inhibition to germination may be not complete by CPC in the presence of sufficient nutrients. Then, the inventors examined the effects by the combination with D-alanine, which is another spore germination inhibitor, and found its remarkable synergistic effects.
In the case of the combination of D-alanine with CPC, a suitable blending amount of CPC, its salt or derivative is 0.01 to 5 wt. % of a food to be stored, preferably 0.05 to 2 wt. % as mentioned previously. A suitable blending amount of D-alanine depends on the L-alanine content of the food to be stored, and is 30 to 200 wt. % of the L-alanine in the food, preferably 40 to 100 wt. %. In general, the blending amount of D-alanine is in the range from 0.01 to 5 wt. % of the food to be stored.
In an embodiment for practicing germination inhibition, CPC, its salt or derivative is added to a food to be stored, and subjected to asteurizaiton to sterilize microorganisms in a vegetative cell state other than spores and to keep the spores in a state of not germinated. The foods treated as above can be inhibited from spoilage and putrefaction by sporogenous bacteria for a long period by a simple storage, compared with foods subjected to only pasteurization. In another embodiment, it is also effective to add CPC, its salt or derivative to food after subjected to pasteurization. In another embodiment, it is also effective to inhibit germination of spores in a state of death or growth inhibition of microorganisms in vegetative cell state other than spores, by a combination of a bacteriocide, fungicide, bacteriostat or fungistat with CPC, its salt or derivative.
Furthermore, it is also effective to apply or spray CPC, its salt or derivative onto the surface of food or the surface of a packaging material which package a food.